Translation of potyvirus,rna in a rabbit reticulocyte lysate: Reaction conditions and identification of capsid protein as one of the products of in vitro translation of tobacco etch and pepper mottle viral RNAs

Abstract

RNAs of pepper mottle virus (PeMV) and tobacco etch virus (TEV) were efficient messengers when translated in the rabbit reticulocyte lysate system. Viral RNA (39 S) isolated from sucrose density gradients stimulated [ 35S]methionine incorporation into products precipitated by trichloroacetic acid 15- to 20-fold over endogenous background levels. Optimal reaction conditions for in vitro translation contained 2 μg RNA/30 μl reaction assay, 0.8–1.0 m M magnesium ions, and 100–125 m M potassium ions. Labeled products from the translation of TEV and PeMV show distinct electrophoretic patterns on sodium dodecyl sulfate polyacrylamide gradient gels (PAGE). The major products of TEV translation had estimated sizes of 87 × 10 4 daltons (87 kd), 85, 54, 49, and 30 kd. PeMV-RNA stimulated the synthesis of 90-, 77-, 68-, 49-, and 33-kd proteins. The 30-kd protein for TEV and the 33-kd protein for PeMV were identified as capsid protein on the basis of estimated size on PAGE, serological reaction, and peptide mapping. The strategy of the in vitro translation of potyvirus RNA is discussed.